This proposal uses an innovative chemoproteomic technology termed isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) to map the druggable hotspots targeted by withaferin A, a natural product derived from Acnistus arborescens that bears a cysteine-reactive Michael acceptor and impairs TNBC cell viability. IsoTOP-ABPP uses reactivity-based chemical probes to map proteome-wide reactive, functional, and druggable hotspots directly in complex proteomes. When used in a competitive manner, covalently-acting small-molecules can be competed against corresponding reactivity-based probes to enable target discovery. Using isoTOP-ABPP platforms, this proposal shows preliminary data for identifying multiple cysteine hotspots targeted by withaferin A, including C377 of PPP2R1A, the regulatory subunit of the major tumor suppressor protein phosphatase 2A (PP2A). The preliminary data show that withaferin A treatment in TNBC cells impairs phosphorylation of the major PP2A substrate and oncogenic signaling protein AKT, potentially explaining its potent anti- cancer activity in these cells. Using fragment-based covalent ligand discovery methods and isoTOP- ABPP platforms, a cysteine-reactive acrylamide lead DKM 2-90 has also been identified that more selectively targets PP2A, impairs phospho-AKT signaling, and impairs TNBC cell viability. This proposal hypothesizes that withaferin A impairs TNBC pathogenicity through targeting a cysteine hotspot in PPP2R1A to activate PP2A activity and impair oncogenic signaling pathways such as AKT. This proposal will use isoTOP-ABPP platforms to investigate the mechanisms of action of withaferin A in impairing TNBC pathogenicity and use covalent ligand discovery approaches to develop more synthetically accessible potent and selective modulators against withaferin A targets.